The molecular basis of modern marker chemistry
von Ruhland, Christopher John
This thesis focuses on empirical investigations and refinements of immunohistochemical marker chemistry to gain insights into the design of novel markers for light and electron microscopy.\ud In Chapter 2, incorporation of d-block metals into polymerised biphenyl-3,3′,4,4′-tetramine (polyDAB) identified complexes of Ni(II), Pt(II), Pt(IV) and Au(III) to be powerful catalysts of silver reduction from physical developers. Na2S(aq) treatment increased the range and activity of catalytic complexes, allowing previously invisible immunohistochemical deposits of polyDAB to be clearly seen in diagnostically relevant samples.\ud Chapter 3 refined this technique by manipulating reagent concentrations whilst suppressing tissue argyrophilia, increasing immunohistochemical sensitivity by an order of magnitude. Marker deposition and thus amplification, was dependent on conjugate quality and coupling method.\ud In Chapter 4, scanning and transmission electron microscopy identified 8 d-block metals that increased the electron opacity of polyDAB, including W(VI), Os(VIII), Pt(II) and Au(III). The majority were detectable by energy dispersive X-ray analysis (EDX), but were present in insufficient quantities for use in analytical electron microscopical tomography (AEMT).\ud In Chapter 5, immunohistochemical polymerisation of halogenated aromatic diamines and bis-diamines as AEMT markers was investigated. The 16 compounds studied produced deposits of varying properties and compositions, morphological criteria identifying those of 1,2-diamino-4-bromobenzene and 1,2-diamino-4,5-diiodobenzene as suitable candidates; EDX indicated that the latter might be applicable to AEMT.\ud Chapter 6 investigated silver deposition from a physical developer by photoconversion. Photo-excitation of immunofluorescently-stained tissue sections in the presence of physical developer caused selective silver deposition at immunopositive sites, a novel method that might find application in AEMT.\ud In Chapter 7, characterisation of polyDAB revealed a molecular weight range of 600 to over 100,000; IR spectra were consistent with an indamine- or phenazine-like polymer. Poor solubility restricted further characterisation.\ud In Chapter 8, additional applications of halogenated compounds were investigated and results suggested potential applications in biological research and diagnosis.
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